Investigating the effect of KRAS gene in human lung cancer using CRISPR-Cas9 system

ABSTRACT

ToolGen Knock-out lentiCRISPRv2 Cloning

SCHEME

Doil Yoon1, Somyung Park1, Yuna Cho1, Jongha Lee1, Wookjin Shin1, Chiyo Mikuni1,2, Jung-uk Lee1,3, Jae-Hyun Lee1,2,* and Jinwoo Cheon1,2,3,*

1Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea 2Graduate Program of Nano Biomedical Engineering (NanoBME), Yonsei-IBS Institute, Yonsei University, Seoul, Republic of Korea

3Department of Chemistry, Yonsei University, Seoul, Republic of Korea

RESULTS

psi+ RRE

LTR LTR

U6 2kb filter sgRNA

scaffold EFS SpCas9 P2A Puro WPRE

cPPT BsmBI BsmBI

lentiCRISPRv2

CONCLUSION & FURTHER STUDY

Fig. 5. lentiCRISPRv2 vector design.

1KB Ladder

lentiCRISPRv2 Enzyme cut

2000 bp

1000 bp

500 bp

3000-10000 bp

REFERENCE

Fig. 6. lentiCRISPRv2 Cloning. (A) lentiCRISPRv2 vector was cut using BsmBI restriction enzyme. sgRNAs were annealed (B) and ligated with lentiCRISPRv2 vector.

A B

Fig. 1. Guide-RNA sequence design. (A) Using guide-RNA evaluation tool from Harvard medical school, lists of potent candidates for guide-RNA are shown. (B) Guide RNAs are selected based on paper and score.

0.0

0.5

1.0

1.5

2.0

N.S. *

*

A549

0.0

0.5

1.0

1.5

2.0

N.S. *

*

H1299

rela

tive

ce

ll d

ensi

ty

rela

tive c

ell

density

significance is determined by unpaired two-tailed Student’s t-test (N.S., no significance; *P < 0.05)

A549 H1299 Control

Cas9-RFP

sgRNA TF

Control

Cas9-RFP

sgRNA TF

A B

Fig. 4. Cell growth rate analysis. (A) Images of cells were obtained using brightfield microscopy. (B) sgRNA transfected cells had decreased cell growth rate.

KRAS guide RNA seq

A549 (Human) CTTGTGGTAGTTGGAGCTAG (TGG–PAM)

MUTANT (G12S)

H1299 (Human) CTTGTGGTAGTTGGAGCTGG (TGG–PAM)

WILD TYPE

Antonia Marazioti, Georgios T. Stathopoulos et al. (2017). ‘Mutant KRAS promotes malignant pleural effusion formation’, Nature Communications, 8, 15205. Colin R. Goding, Jun Zhou, Rutao Cui et al. (2019). ‘Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads’, Nature Communications, 10, 877. David Barras. (2015). ‘BRAF Mutation and Its Importance in Colorectal Cancer’, Biomarkers in Cancer, 7, 9-12. Davies H et al. (2002). ‘Mutations of the BRAF gene in human cancer’, Nature, 417(6892), 949-54. Haiwei Mou, Jill Moore, Sunil K. Malonia, Yingxiang Li, Deniz M. Ozata, Soren Hough, Chun-Qing Song, Jordan L. Smith, Andrew Fischer, Zhiping Weng, Michael R. Green, Wen Xue. (2017). ‘Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis’, Proceedings of the National Academy of Sciences of the United States of America, 114(14), 3648-3653. Patrick D. Hsu, Eric S. Lander, Feng Zhang. (2014). ‘Development and Applications of CRISPR-Cas9 for Genome Engineering’, Cell, 157(6), 1262-78. Wookjae Lee, Joon Ho Lee, Soyeong Jun, Ji Hyun Lee, Duhee Bang. (2018). ‘Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells’, Scientific Reports, 8, 11879.

Lung cancer, also known as lung carcinoma, is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. It is known to be difficult to detect on early stages, and commonly spreads to other tissues. Currently available treatments usually targets the EGF(Epidermal growth factor) receptor. However, recent studies show that the EGF receptor targeting molecules have low efficiency - which might be due to the mutations on downstream signaling molecules. In this study, we knocked out important signaling kinase, KRAS, and identified its role on cell growth rate. When we knocked out the KRAS gene, a significant decrease on cellular growth rate was observed, regardless of the existence of mutations on the KRAS gene. Our data might provide a new stepping stone into the development of new lung cancer drug targeting KRAS in the future, as it could indicate a potentially more efficient method in decreasing the growth level of tumor cells in lung tissues.

A B

PAM

CTTGTGGTAGTTGGAGCTAG

Guide RNA

Target DNA 5’

3’ 5’

3’

Cas9

U6 sgRNA CMV

sgRNA expression vector

RFP CAS9 CMV

Cas9-RFP expression vector

Transfection

Strategy

I. ToolGen Knock-out II. lentiCRSIPRv2 Cloning

Key Concept

5’ 3’

3’ 5’

Anneal

Digest

BsmBI LentiCRISPR V2

Cas9

sgRNA Scaffold

LentiCRISPR V2 Ligate

LentiCRISPR V2

Cas9

sgRNA

Cas9

sgRNA Oligo Annealed sgRNA

Ligands

EGFR Cell Membrane

PI3K KRAS

AKT

BRAF

MEK/ERK

Nucleus

Proliferation Survival

Angiogenesis Migration

PTEN

Knock-out

No Treat (Wild type) Hypothesis

Growth rate change?

CRIAPR/Cas9

A549 H1299

A549

H1299

Control Cas9-RFP sgRNA TF

mRNA

β-Actin

β-Actin

K-Ras

K-Ras

Control Cas9-RFP sgRNA TF

Protein level

Fig. 3. Comparison of mRNA and protein level. RT-PCR and PCR were done to compare mRNA level. Western blot analysis was used to check the protein level. K-Ras bands were replaced by a drawing since antibody for K-Ras shows low specificity.

Our team conducted a total of three experiments to confirm successful knock out using CRISPR-Cas9. Because of the low efficiency of antibodies and property of KRAS gene itself, experiments that directly check the amount of mRNA and protein did not produce satisfactory results. However, in the process of counting the number of cells and confirming their growth rate, a significant difference was observed. Later, a quantitative experiment will be conducted to confirm the decrease in growth rate. Also, by knocking in the KRAS gene again and seeing whether the growth rate is restored, we could solidify our results. Cloned lentiCRISPRv2 that we made for this study might use for knock-out experiment for further studies. This might increase the chance to make efficient KRAS knocked out human lung cancer cell line library. If knocking out the KRAS gene yields a significant decrease in tumor cell growth rate, further research into developing drugs targeting the KRAS gene might be possible.

A549 H1299

Day 0

Day 1

Day 7

BF RFP (Cas9) Merged

Selection

Day 0

Day 6

Day 8

Day 10

BF RFP (Cas9) Merged

Selection

A B

Fig. 2. Cas9 transfection and selection. Cas9-RFP vector was transfected into each cell line (A549–A, H1299-B) and grown under the condition of Puromycin.

Day 18